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Akt regulates glutamate receptor trafficking and postsynaptic membrane elaboration at the Drosophila neuromuscular junction.

Lee HG, Zhao N, Campion BK, Nguyen MM, Selleck SB - Dev Neurobiol (2013)

Bottom Line: The single Drosophila Akt family member, Akt1 selectively altered the postsynaptic targeting of one glutamate receptor subunit, GluRIIA, and was required for the expansion of a specialized postsynaptic membrane compartment, the subsynaptic reticulum (SSR).Several lines of evidence indicated that Akt1 influences SSR assembly by regulation of Gtaxin, a Drosophila t-SNARE protein (Gorczyca et al., 2007) in a manner independent of the mislocalization of GluRIIA.Our findings show that Akt1 governs two critical elements of synapse development, neurotransmitter receptor localization, and postsynaptic membrane elaboration.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, 16802.

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Influence of Akt1 on Dorsal and Cactus levels and distribution at the NMJ. A and B: In control animals (UAS-Akt1RNAi/+), Dorsal (detected by anti-Dorsal antibody; red) is localized to the postsynaptic specialization. Neuronal projections were labeled by anti-HRP staining (green). C and D: Akt1 function was compromised by expressing UAS-Akt1RNAi under the muscle-specific 24B-GAL4. Dorsal levels at the NMJ were significantly reduced. E and F: Cactus (red) was concentrated at the NMJ in control animals. G and H: Inhibition of Akt1 function in the muscle (24B-GAL4/UAS-Akt1RNAi) resulted in reduced levels of Cactus at the NMJ. I–K: In control animals, Dorsal immunoreactivity (red) colocalized with GluRIIA (green) immunoreactivity at the postsynaptic specialization. L–N: In muscles where Akt1 expression was inhibited, both Dorsal and GluRIIA redistributed into intracellular bands, although the effect on Dorsal was less and incompletely penetrant. Mislocalized Dorsal partially overlapped with GluRIIA (arrowheads indicate bands of Dorsal and GluRIIA; arrows indicate synaptic boutons). Scale bar in (A–H), 50 µm, in (I–N), 5 µm.
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fig04: Influence of Akt1 on Dorsal and Cactus levels and distribution at the NMJ. A and B: In control animals (UAS-Akt1RNAi/+), Dorsal (detected by anti-Dorsal antibody; red) is localized to the postsynaptic specialization. Neuronal projections were labeled by anti-HRP staining (green). C and D: Akt1 function was compromised by expressing UAS-Akt1RNAi under the muscle-specific 24B-GAL4. Dorsal levels at the NMJ were significantly reduced. E and F: Cactus (red) was concentrated at the NMJ in control animals. G and H: Inhibition of Akt1 function in the muscle (24B-GAL4/UAS-Akt1RNAi) resulted in reduced levels of Cactus at the NMJ. I–K: In control animals, Dorsal immunoreactivity (red) colocalized with GluRIIA (green) immunoreactivity at the postsynaptic specialization. L–N: In muscles where Akt1 expression was inhibited, both Dorsal and GluRIIA redistributed into intracellular bands, although the effect on Dorsal was less and incompletely penetrant. Mislocalized Dorsal partially overlapped with GluRIIA (arrowheads indicate bands of Dorsal and GluRIIA; arrows indicate synaptic boutons). Scale bar in (A–H), 50 µm, in (I–N), 5 µm.

Mentions: We also examined the effect of Akt1 on two potential downstream targets, Dorsal and Cactus (Drosophila homologs of NF-κB and Iκ-B, respectively). These two proteins have recently been shown to localize to postsynaptic specializations and regulate glutamate receptor levels at the NMJ (Heckscher et al., 2007). While Dorsal and Cactus have been well characterized as transcriptional activator proteins, their activity at the NMJ is posttranscriptional, affecting the localization or stabilization of glutamate receptors in the SSR (Heckscher et al., 2007). To determine whether Akt1's effects on GluRIIA localization could be mediated at least in part by an influence on Dorsal or Cactus, the levels and distributions of these two proteins at the NMJ were evaluated. As previously described, Dorsal and Cactus were concentrated in postsynaptic specializations at type Ib boutons in control animals [Fig. 4(A,B,E,F)]. Upon RNAi knockdown of Akt1, both Dorsal and Cactus levels significantly decreased at the NMJ [Fig. 4(C,D,G,H)] [Supporting Information Fig. 1(B)]. In addition to the reduction of Dorsal levels at the NMJ, Dorsal was mislocalized in a number of animals (23.8% penetrance) and partially colocalized with GluRIIA into intracellular bands in the muscle cell [Fig. 4(L–N); arrowheads indicate the bands of GluRIIA and Dorsal, arrows indicate synaptic boutons]. Although the penetrance of this phenotype was modest, it was reproducible across three different sets of experiments. These findings showed that Akt1 affects the levels of two potential Akt1 downstream targets known to regulate GluRIIA levels, and suggest the possibility that Akt1 regulates GluRIIA at least in part via the control of Dorsal and Cactus.


Akt regulates glutamate receptor trafficking and postsynaptic membrane elaboration at the Drosophila neuromuscular junction.

Lee HG, Zhao N, Campion BK, Nguyen MM, Selleck SB - Dev Neurobiol (2013)

Influence of Akt1 on Dorsal and Cactus levels and distribution at the NMJ. A and B: In control animals (UAS-Akt1RNAi/+), Dorsal (detected by anti-Dorsal antibody; red) is localized to the postsynaptic specialization. Neuronal projections were labeled by anti-HRP staining (green). C and D: Akt1 function was compromised by expressing UAS-Akt1RNAi under the muscle-specific 24B-GAL4. Dorsal levels at the NMJ were significantly reduced. E and F: Cactus (red) was concentrated at the NMJ in control animals. G and H: Inhibition of Akt1 function in the muscle (24B-GAL4/UAS-Akt1RNAi) resulted in reduced levels of Cactus at the NMJ. I–K: In control animals, Dorsal immunoreactivity (red) colocalized with GluRIIA (green) immunoreactivity at the postsynaptic specialization. L–N: In muscles where Akt1 expression was inhibited, both Dorsal and GluRIIA redistributed into intracellular bands, although the effect on Dorsal was less and incompletely penetrant. Mislocalized Dorsal partially overlapped with GluRIIA (arrowheads indicate bands of Dorsal and GluRIIA; arrows indicate synaptic boutons). Scale bar in (A–H), 50 µm, in (I–N), 5 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig04: Influence of Akt1 on Dorsal and Cactus levels and distribution at the NMJ. A and B: In control animals (UAS-Akt1RNAi/+), Dorsal (detected by anti-Dorsal antibody; red) is localized to the postsynaptic specialization. Neuronal projections were labeled by anti-HRP staining (green). C and D: Akt1 function was compromised by expressing UAS-Akt1RNAi under the muscle-specific 24B-GAL4. Dorsal levels at the NMJ were significantly reduced. E and F: Cactus (red) was concentrated at the NMJ in control animals. G and H: Inhibition of Akt1 function in the muscle (24B-GAL4/UAS-Akt1RNAi) resulted in reduced levels of Cactus at the NMJ. I–K: In control animals, Dorsal immunoreactivity (red) colocalized with GluRIIA (green) immunoreactivity at the postsynaptic specialization. L–N: In muscles where Akt1 expression was inhibited, both Dorsal and GluRIIA redistributed into intracellular bands, although the effect on Dorsal was less and incompletely penetrant. Mislocalized Dorsal partially overlapped with GluRIIA (arrowheads indicate bands of Dorsal and GluRIIA; arrows indicate synaptic boutons). Scale bar in (A–H), 50 µm, in (I–N), 5 µm.
Mentions: We also examined the effect of Akt1 on two potential downstream targets, Dorsal and Cactus (Drosophila homologs of NF-κB and Iκ-B, respectively). These two proteins have recently been shown to localize to postsynaptic specializations and regulate glutamate receptor levels at the NMJ (Heckscher et al., 2007). While Dorsal and Cactus have been well characterized as transcriptional activator proteins, their activity at the NMJ is posttranscriptional, affecting the localization or stabilization of glutamate receptors in the SSR (Heckscher et al., 2007). To determine whether Akt1's effects on GluRIIA localization could be mediated at least in part by an influence on Dorsal or Cactus, the levels and distributions of these two proteins at the NMJ were evaluated. As previously described, Dorsal and Cactus were concentrated in postsynaptic specializations at type Ib boutons in control animals [Fig. 4(A,B,E,F)]. Upon RNAi knockdown of Akt1, both Dorsal and Cactus levels significantly decreased at the NMJ [Fig. 4(C,D,G,H)] [Supporting Information Fig. 1(B)]. In addition to the reduction of Dorsal levels at the NMJ, Dorsal was mislocalized in a number of animals (23.8% penetrance) and partially colocalized with GluRIIA into intracellular bands in the muscle cell [Fig. 4(L–N); arrowheads indicate the bands of GluRIIA and Dorsal, arrows indicate synaptic boutons]. Although the penetrance of this phenotype was modest, it was reproducible across three different sets of experiments. These findings showed that Akt1 affects the levels of two potential Akt1 downstream targets known to regulate GluRIIA levels, and suggest the possibility that Akt1 regulates GluRIIA at least in part via the control of Dorsal and Cactus.

Bottom Line: The single Drosophila Akt family member, Akt1 selectively altered the postsynaptic targeting of one glutamate receptor subunit, GluRIIA, and was required for the expansion of a specialized postsynaptic membrane compartment, the subsynaptic reticulum (SSR).Several lines of evidence indicated that Akt1 influences SSR assembly by regulation of Gtaxin, a Drosophila t-SNARE protein (Gorczyca et al., 2007) in a manner independent of the mislocalization of GluRIIA.Our findings show that Akt1 governs two critical elements of synapse development, neurotransmitter receptor localization, and postsynaptic membrane elaboration.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, 16802.

Show MeSH
Related in: MedlinePlus